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Regular Article |
a Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, gene regulation, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure of testes to ionizing radiation efficiently induces apoptosis of germ cells, with the actively dividing spermatogonia being the most susceptible, followed by stem cell spermatogonia, spermatocytes, and the highly resistant spermatids (for a summary of relevant work, see [5]). Germ cell death resulting from radiation occurs via apoptosis [3, 6]. On the other hand, the supportive, nondividing Sertoli cells are highly resistant to radiation. Although p53 is necessary for radiation-induced germ cell death [7], the downstream events following p53 activation in testis have not been explored. The Fas system is one of several pathways that have been implicated in p53-dependent cell death.
Fas is a cell surface receptor that, on ligand binding (Tnfsf6/FasL/CD95L), activates rapid apoptosis via a caspase cascade initiated by caspase 8. Most of what is known regarding Fas comes from the immune system, because Fas/FasL interactions play a clear role in thymic cell deletion, natural killer cell and activated T-cell function, and peripheral T-cell deletion. Although it is accepted that the Fas system is also present in the testis, the roles that it plays in this tissue are not as well defined. Fas/FasL interactions are proposed to be important in maintaining the immune-privilege status of the testis [8], in germ cell development [9], in spermatozoal survival in the female genital tract [10], and in responses to injury [11, 12]. Fas receptor has been localized to the germ cells, whereas FasL expression has been localized to the Sertoli cells [9, 12].
The nature of the interaction between p53 and Fas in the execution of cell death remains elusive. However, evidence suggests that Fas is activated downstream of p53 both by transcriptional up-regulation and by regulation at the protein level. Fas mRNA is up-regulated in many cell types following p53 induction [13–15], and in vitro, both mouse and human Fas induction are dependent on a p53-response element in the first intron [16]. Additionally, evidence for p53 regulation of Fas at the protein level comes from experiments demonstrating that p53 induction leads to trafficking of Fas from preformed pools in the Golgi apparatus [17].
Most of the work defining p53 and Fas/FasL interactions has been performed in vitro and has involved cells from the immune system. The role of Fas in the response to radiation has been defined by studies in tumor cells and hematopoietic cell types [13, 18, 19]. However, to our knowledge, the role of Fas in radiation-induced testicular germ cell apoptosis has not been examined previously. Here, using an in vivo system, the possibility that Fas is involved in p53-dependent germ cell death is explored.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice of a mixed background were bred and maintained in an in-house colony. The colony was derived from an initial stock of five p53-/- male mice (C57BL/6J-Trp53tm1Tyj) and 15 gld female mice (B6Smn.C3H-Tnfsf6gld) from The Jackson Laboratory (Bar Harbor, ME). All B6 wild-type, p53-/-, and gld mice were derived from crosses of the F1 offspring. Genotyping was done using multiplex polymerase chain reaction (PCR) followed by restriction digestion. Lprcg (CBA/KlJms-Tnfrsf6lpr-cg) and control CBA (CBA/J) mice were ordered from The Jackson Laboratory and were acclimated in-house for 1 wk before treatment. All mice were 8 wk old at the time of radiation exposure. Animals were housed in humidity (30%–70%)- and temperature (74 ± 2°F)-controlled rooms and maintained on a 12L:12D photoperiod. Animals had access to Purina Rodent Chow 5001 (Farmers Exchange, Framingham, MA) and water ad libitum. All procedures involving animals were performed in accordance with the National Research Council's Guide for Care and Use of Laboratory Animals and the guidelines of Brown University's Institutional Animal Care and Use Committee.
Genotyping
The DNA was isolated from tail tips, which were collected from 21- to 28-day-old mice while the mice were anesthetized with Metophane (inhalation) (Schering-Plough, Union, NJ). Template DNA (25 ng) was used for a 50-µl multiplex PCR reaction containing 200 µM dNTPs (Gibco, Gaithersburg, MD), 1x PCR buffer (Perkin Elmer, Branschburg, NJ), 6 mM MgCl2 (Perkin Elmer), 0.05 U/µl of AmpliTaq Gold (Perkin Elmer), 0.2 µM of each FasL primer (5'-ATAGGTCTTAAGAAGACTCTCATTCAAG-3' and 5'-TGATCAATTTTGAGGAATCTAAGGCC-3'), 0.2 µM of each neomycin-resistance primer (5'-AGGTGAGATGACAGGAGATC-3' and 5'-CTTGGGTGGAGAGGCTATTC-3'), and 0.2 µM of p53 primer (5'-GCGTCTTAGAGACAGTTGACT-3', and 5'-GGATAGGTCGGCGGTTCATGC-3') in a Perkin Elmer 2400 Thermocycler as follows: 95°C for 10 min; followed by 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and ending with 72°C for 5 min. To identify the presence or absence of the FasL mutation in gld mice, a restriction digest was performed using 15 µl of the PCR product, 1 µl of StuI (Gibco), and 0.5 µl of 1 M NaCl2. The samples were incubated for 3 h at 37°C, followed by 10 min at 65°C. The resulting product was separated on a 2.5% (w/v) agarose gel, and the presence or absence of bands at 112, 136, 278, and 458 base pairs (bp) indicated the presence of a wild-type FasL allele, a gld mutant FasL allele, a p53 knock-out allele, and a p53 wild-type allele, respectively.
Irradiation and Experimental Outline
Unanesthetized, 8-wk-old male mice were exposed to radiation with single doses of 0.5 or 5.0 Gy at a rate of 1.01–1.67 Gy/min using a Philips 250-kVp x-ray machine. Dose rate was calculated using a Victoreen probe. Animals were restrained in polystyrene chambers, and the upper two-thirds of the body were shielded with 3 mm of lead. At designated time points, animals were killed by CO2 asphyxiation, and testes were excised immediately. In animals dosed with 0.5 Gy of radiation, both testes were flash-frozen and stored at -80°C until analysis for spermatid head counts. For animals dosed with 5 Gy of radiation, the right testis was flash-frozen for RNA analysis, and the left testis was flash-frozen in Tissue-Tek O.C.T. Compound (Sakura Finetek, Torrance, CA) for cryosectioning and in situ TUNEL staining. Frozen samples were stored at -80°C until analysis.
Semiquantitative Reverse Transcription-PCR
The RNA was extracted from frozen whole testes using the Tri reagent (Molecular Research Center, Cincinnati, OH). First-strand cDNA was made using 1 µg of total RNA in the presence of Superscript II reverse transcriptase (Gibco/BRL) and random hexamers. The reverse-transcribed mixture (1 µl) was used as template for subsequent PCR reactions. A previously described method of semiquantitative PCR [20] was used to monitor mRNA expression of Fas and FasL. Briefly, PCR products of mouse Fas (331 bp), FasL (166 bp), and ß-actin (429 bp) were amplified using primers for Fas (5'-GAGAATTGCTGAAGACATGACAATCC-3' and 5'-ATGGCTGGAACTGAGGTAGTTTTCAC-3'), FasL (5'-TTATATGTCAACATATCTCAACTCTCTCTG-3' and 5'-ATAGGTCTTAAGAAGACTCTCATTCAAG-3'), and ß-actin (5'-AGGAATCCTGACCCTGAAGTACC-3', and 5'-AGCTGTGGTGGTGAAGCTGTAGC-3'). To test for the presence of any contaminating DNA, negative-control reactions, without reverse transcriptase, were run concomitantly with cDNA samples. For quantitative analysis, ß-actin was coamplified in each reaction as an internal control. The PCR products (10 µl) were collected during each cycle between cycles 25 and 32 (Fas) or cycles 27 and 34 (FasL) and were run out on a 2.5% (w/v) agarose gel. The image of the gel was captured on a Gel Doc 2000 using accompanying Quantity 1 version 4.0.1 software (Bio-Rad, Hercules, CA), and the intensity of ethidium bromide staining of the bands was analyzed using NIH Image (Bethesda, MD). The relative ratio of Fas or FasL to ß-actin was calculated from cycles in which the amplification of both PCR products was exponential. Data were normalized to the within-run, wild-type, 0-h time point to account for potential differences in PCR master mix preparations or reverse transcription reactions from day to day.
Detection of Apoptosis
Germ cell apoptosis was detected in 8-µm cryosections from frozen testes by TUNEL staining using ApopTag kits (Intergen, Purchase, NY). Tissue was counterstained with methyl green. Each tissue section was examined for TUNEL-positive cells, and the apoptotic index was determined by the ratio of the number of essentially round seminiferous tubules with more than 3 TUNEL-positive cells relative to the total number of essentially round tubules. For each testis section, 100–200 seminiferous tubules were counted.
Low Molecular Weight DNA Isolation
The presence of a low molecular weight ladder was used to verify that radiation-induced cell death was apoptosis. Twelve hours after exposure to 5 Gy radiation, testes were decapsulated, washed in Eagle media, and frozen in liquid nitrogen. Frozen tissue was homogenized in homogenization buffer (5 mM Tris-HCl, 20 mM EDTA, and 0.5% (v/v) Triton X-100, pH 8) in a Dounce homogenizer and incubated with diethyl pyrocarbonate for 10 min. The homogenate was centrifuged at 16 000 x g at 4°C for 40 min. The supernatent was incubated with 0.5 mg/ml of proteinase K at 37°C for 1 h and extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1; v/v/v). The aqueous phase was ethanol-precipitated overnight, pelleted, and resuspended in TE buffer (pH 8) containing 10 mM Tris-HCl and 1 mM EDTA. The resulting DNA was digested with RNase free of DNase at 37°C for 1 h and was extracted. The aqueous phase was ethanol-precipitated overnight with 20 µg/ml of glycogen, pelleted, and resuspended in TE buffer. A total of 3 µg of DNA were loaded on a 2% (w/v) agarose gel and separated by electrophoresis. The DNA was stained with ethidium bromide and visualized with an ultraviolet transilluminator. The sizes of the resulting DNA bands were estimated by comparison with a standard 100-bp ladder.
Spermatid Head Counts
Testes obtained 29 days after irradiation and from unirradiated controls were stored at -80°C until analysis. Both testes from each animal were homogenized separately, and sperm heads were counted on a hemocytometer using previously described methods [21]. The counts from the two testes of each animal were averaged for statistical analysis.
Statistics
The mean and SEM were calculated for each data point and are presented in the figures. The apoptotic index and spermatid head counts were analyzed by ANOVA followed by a Fisher protected least significant difference test using Statview 4.0 statistical software (SAS, Cary, NC). Fas and FasL mRNA expression were analyzed with cross-sectional time-series analysis with a generalized estimating equation using STATA 7.0 statistical software (Stata Corporation, College Station, TX).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In mice, genetic background can significantly alter the germ cell response to radiation [22]. Therefore, to verify that our mice, from a mixed genetic background, responded to radiation similarly to those in previous reports, B6 wild-type and p53 knock-out mice were exposed to 5 Gy of x-radiation. In C57BL/6 mice, this dose is known to induce apoptosis in spermatogonia and spermatocytes [6], and the absence of p53 protects germ cells from radiation-induced apoptosis [7]. Animals were killed at 0, 3, 6, 9 and 12 h following radiation to evaluate TUNEL-labeled cells (Fig. 1, a and b). Apoptosis, as assessed by counting seminiferous tubules with more than three apoptotic cells, was increased at 9 and 12 h following radiation in B6 wild-type, but not p53 knock-out, testes (Fig. 1c). Apoptosis was verified by the display of a low molecular weight DNA laddering in B6 wild-type, but not p53 knock-out, mouse testes 12 h following radiation (Fig. 1d). Gld mice, deficient in FasL, also showed a low molecular weight DNA laddering in response to radiation (Fig. 1d); the apoptotic response of gld mice to radiation injury is defined further below.
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Based on these results and those of previous studies [7], the 12-h time point was chosen to quantitate apoptosis after exposure to 5 Gy of x-radiation. The number of apoptotic seminiferous tubules increased significantly in B6 wild-type animals 12 h following radiation, whereas apoptosis in p53 knock-out testes remained near normal, verifying that radiation-induced germ cell apoptosis is p53-dependent in these mice (mean apoptotic index ± SEM was 0.53 ± 0.063 in wild-type mice and 0.04 ± 0.021 in p53 knock-out mice; n = 4 mice/genotype; P < 0.01 by ANOVA and Fisher protected least significant difference test).
Survival and Differentiation of A1 to B Spermatogonia Following Exposure to 0.5 Gy of X-Radiation
To examine the ability of A1 through B spermatogonia to survive and differentiate into late spermatids, homogenization-resistant spermatid head counts were evaluated 29 days after 0.5 Gy of x-radiation. Historical control data from the p53 knock-out and B6 wild-type male mice from our lab, as well as data from a previously published report [7], indicate that spermatid head counts in p53 knock-out and B6 wild-type testes are similar and range from 17.5 to 20 million sperm per testis. Consistent with the analysis at 12 h after exposure to 5 Gy of x-radiation, B6 wild-type mice showed an approximately 60% reduction in numbers of spermatid heads per testis, whereas p53-deficient mice had near-normal levels of spermatid heads (Fig. 2).
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Fas/FasL Expression Following Exposure to 5 Gy of X-Radiation
To examine whether Fas is an apoptotic mechanism induced by p53, Fas expression was examined using semiquantitative reverse transcription-PCR. Fas mRNA was increased 3 h after exposure to 5 Gy of x-radiation (Fig. 3a). This increase in Fas mRNA was p53-dependent, indicating that increased Fas expression is downstream of p53 and might be part of the p53-dependent apoptosis that occurs following radiation. Interestingly, FasL expression, as determined by reverse transcription-PCR, remained unchanged following radiation (Fig. 3b). The levels of Fas and FasL mRNA were also measured in the testes of gld mice (n = 1 animal/time point) following radiation, and similar to B6 wild-type mice, an increase in Fas, but not in FasL, expression was observed following radiation (data not shown).
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Effects of Functional Elimination of Fas or FasL on Response to Radiation
To determine the functional significance of Fas expression in the context of radiation injury, lprcg mice, having a nonfunctional Fas, were exposed to 5 Gy of x-radiation, and testes were examined at 12 h following radiation for apoptosis. Lprcg mice express Fas mRNA of normal size and as abundantly as wild-type mice, but the mRNA carries a point mutation that prevents Fas from transducing an apoptotic signal [23].
A dose of 5 Gy induced apoptosis in both lprcg and CBA wild-type mice (Fig. 4a). However, mice with the lprcg mutation had significantly lower levels of apoptosis at 12 h following radiation as compared to CBA wild-type controls. Lprcg mice had a 203% increase in apoptosis compared to unexposed lprcg mice, whereas CBA wild-type mice had a 428% increase in apoptosis compared to unexposed controls.
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Spermatid head counts from testes 29 days after exposure to 0.5 Gy were consistent with the effects on apoptosis (Fig. 4b). Spermatid head counts were significantly higher in lprcg mice exposed to 0.5 Gy of radiation when compared to treated CBA wild-type mice. Spermatid head counts in CBA wild-type mice were reduced by 72% after exposure to 0.5 Gy of radiation, whereas spermatid head counts were reduced by 56% in lprcg mice.
Interestingly, when gld mice, containing a point mutation in FasL, were exposed to 5 Gy of x-radiation, apoptosis was induced to levels similar to those of treated B6 wild-type animals (Fig. 5a). Similarly, when gld mice were exposed to 0.5 Gy of radiation, spermatid head counts were reduced to levels equivalent to those of treated B6 wild-type mice (Fig. 5b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fas, a receptor that induces apoptosis on activation, was transcriptionally up-regulated following ionizing radiation, and this increase was dependent on the presence of functional p53. Consistent with functional significance of the up-regulation of Fas was the timing of the increase (3 h following radiation) relative to the timing of apoptosis (9–12 h following radiation). Germ cells in lprcg mice, lacking functional Fas, were partially resistant to ionizing radiation. However, this resistance was not as extensive as that when p53 was eliminated.
The up-regulation of Fas mRNA in a timely manner, the dependence of Fas expression on the presence of p53, and the protection conferred by a mutation in Fas all suggest that Fas plays an important role in the radiation response. However, the lack of full protection against radiation in the lprcg mouse indicates that Fas may be one of several apoptotic mechanisms induced by p53 and that, in the absence of Fas, other pathways still induce some apoptosis.
Interestingly, germ cells of gld mice, having a nonfunctional FasL, were sensitive to radiation-induced apoptosis, whereas lprcg mice, with a nonfunctional Fas, were somewhat protected from radiation. These results indicate that, although Fas is important in the apoptotic response to radiation, the presence of FasL is not necessary. Mechanisms to explain these observed differences include that Fas is activated in response to radiation in the absence of a ligand or that an alternate ligand to FasL exists. Previous reports have shown that Fas can be activated directly by ultraviolet radiation in the absence of ligand [24]. Additionally, evidence suggests that Fas may be activated by tumor necrosis factor 
(TNF) in the absence of FasL [25]. Given that macrophages increase TNF in response to radiation [26], it is possible that resident macrophages secrete enough TNF to activate Fas. Alternatively, small amounts of TNF have been found to be expressed in round spermatids and pachytene spermatocytes in adult mouse testis [27]. Exposure to radiation might induce higher expression of TNF in spermatids and spermatocytes—high enough to engage Fas on germ cells. Interestingly, prolonged exposure to high levels of TNF induces cell death in both spermatocytes and spermatids [28].
Differences in the strains of mice also may have contributed to different responses to radiation, because the gld mice were of a mixed B6 background and the lprcg mice were of a CBA background. Variation in modifying proteins or even transcription factors in the strains of mice (C57BL/6 vs. CBA) could allow an effect to be observed in one strain and not the other. Alternatively, a polymorphism in Fas or FasL could lead to differences in the role of Fas/FasL interactions depending on background. Regardless of the explanation for the differences seen between gld and lprcg mice, the data are consistent with Fas involvement in the response to radiation.
These observations, in fact, are consistent with a model in which germ cells, but not Sertoli cells, are the target of radiation injury. Spermatogonia and early pachytene spermatocytes up-regulate p53 within 3 h following radiation [29, 30] and undergo apoptosis by a p53-dependent mechanism [7]. Given this cell specific up-regulation of p53 and the data indicating that Fas can be up-regulated by p53 in other systems [13–17], it is not unreasonable to imagine that Fas, although normally expressed by spermatocytes [9], would be up-regulated by p53 in spermatogonia and early pachytene spermatocytes following radiation injury. Indeed, that the lprcg mice showed partial resistance to radiation 29 days following radiation indicates that Fas plays a role in spermatogonial apoptosis, because this is the population affected by the low dose (0.5 Gy) of radiation and that develops into elongate spermatids 29 days following radiation. So, although Fas is expressed on spermatocytes during normal homeostasis [9], Fas likely is induced in spermatogonia following radiation. On the other hand, Sertoli cells constitutively express a low level of FasL, presumably as part of a mechanism to eliminate excess germ cells from the seminiferous epithelium [9]. Our data indicate that the levels of FasL expression are not altered following radiation, and that FasL is not necessary for apoptosis following radiation injury.
Thus, we propose a model in which radiation induces DNA damage within the proliferating spermatogonia and spermatocytes, and in which p53, an intracellularly activated apoptotic protein, is stabilized within germ cells in response to the injury. This, in turn, leads to increased transcription of Fas within the germ cells. Fas expression on cells that do not normally express Fas (spermatogonia) or increased expression on cells that normally express Fas at low levels increases the susceptibility of the germ cells to apoptosis, whether it be through binding of constitutively expressed FasL or of an alternate ligand. Although Fas is traditionally thought of as a receptor for extracellularly activated apoptosis, in this model Fas serves to enhance an intracellularly activated pathway.
Although categorizing pathways into intracellularly versus extracellularly activated makes them easier to understand conceptually, these two categories may not be mutually exclusive, and pathways that traditionally are thought of as being activated by extracellular signals may, in fact, be integrated into the pathways that are activated by intracellular sensors. In the apoptotic response to radiation in testis, we found that p53, an intracellular sensor, induced Fas expression, which is traditionally thought of as an extracellular sensor, and that FasL was not necessary for the induction of apoptosis. Whereas a mutant Fas receptor protected germ cells from radiation-induced cell death, a mutant Fas ligand had no effect, indicating that the Fas-expressing germ cells are able to activate the Fas system in the absence of FasL. This suggests that the Fas system should not be considered as exclusively part of the extracellular sensory pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIEHS grant RO1-ES05033 and by the Burroughs Wellcome Fund. 
2 Correspondence: Kim Boekelheide, Department of Pathology and Laboratory Medicine, Box G-B5, 171 Meeting Street, Brown University, Providence, RI 02912. FAX: 401 863 9008; kim_boekelheide{at}brown.edu
Accepted: December 10, 2001.
Received: September 7, 2001.
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acts as a ligand for Fas in vaginal cells. Cell Death Differ 1999; 6:638-643[CrossRef][Medline] by murine peritoneal macrophages in response to irradiation. Radiat Res 1994; 139:103-108[CrossRef][Medline] in mouse spermatogenic cells. Endocrinology 1993; 133:389-396[Abstract]This article has been cited by other articles:
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  G. Shetty, S. H. Shao, and C. C. Y. Weng p53-Dependent Apoptosis in the Inhibition of Spermatogonial Differentiation in Juvenile Spermatogonial Depletion (Utp14bjsd) Mice Endocrinology, June 1, 2008; 149(6): 2773 - 2781. [Abstract] [Full Text] [PDF]
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 J. S. Moffit, K. Boekelheide, J. M. Sedivy, and J. Klysik Mice Lacking Raf Kinase Inhibitor Protein-1 (RKIP-1) Have Altered Sperm Capacitation and Reduced Reproduction Rates With a Normal Response to Testicular Injury J Androl, November 1, 2007; 28(6): 883 - 890. [Abstract] [Full Text] [PDF]
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T. Rasoulpour, K. DiPalma, B. Kolvek, and M. Hixon Akt1 Suppresses Radiation-Induced Germ Cell Apoptosis in Vivo Endocrinology, September 1, 2006; 147(9): 4213 - 4221. [Abstract] [Full Text] [PDF]
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) versus p53 knock-out (dashed line, •) mice (n = 1 animal/time point/genotype). d) Low molecular weight DNA isolated 12 h post-dose from p53 knock-out, gld, or B6 wild-type mouse testes unexposed (0 Gy) or exposed (5 Gy) to radiation. Each lane represents DNA isolated from a single testis. Standard lane represents 100-bp ladder
